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ABSTRACT:

ABSTRACT:

conlan
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ABSTRACT:

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  1. ABSTRACT: We are interested in determining the conformational changes induced by ligand binding in the intracellular lipid binding protein (iLBP) karitinocyte fatty acid binding protein (K-FABP). The source of this interest is the differential behavior of K-FABP when ligand bound. If it binds a non-activating ligand, such as stearic acid, K-FABP acts as a typical fatty acid binding protein, chaperoning the ligand in the aqueous environment of the cytosol. If, however, K-FABP binds an activating ligand such as linolenic acid, the protein is directed to the nucleus of the cell. The source of this differential behavior is proposed to be the formation of a non-linear nuclear localization sequence (NLS) through conformational changes induced by the binding of an activating ligand. By determining the structure of K-FABP in both the activated and non-activated states we will be able to understand the basis for this curious behavior.

  2. Nuclear localization Subcellular targeting of a protein to the nucleus via a NLS “classical” NLS K(K/R)X(K/R) Such an NLS is recognizable by adaptor proteins called -importins that subsequently interact with -importins to control nuclear localization. Three iLBPs enhance transcriptional activity of nuclear receptors with which they share a common ligand: CRABP II RAR A-FABP PPAR/ K-FABP PPAR

  3. Problem: None of these iLBPs contains a NLS Furthermore… Nuclear localization only occurs upon binding of ligand

  4. CRABP II story Retinoic acid (RA) induces nuclear import of CRABP II Nuclear export signal (NES) MDLCQAFSDVILAEF Leptomycin B (LMB) inhibits NES mediated export COS-7 cells transfected with denoted CRABP II expression vectors (Sessler & Noy 2005)

  5. CRABP II story In the absence of a NLS, a conformational change upon RA binding must “create” a non-linear NLS RA binding induces a basic patch at the end of helix 2 Resulting in a topology for K20, R29 and K30 that mimics a NLS SV40 NLS peptide (Sessler & Noy 2005)

  6. CRABP II story colored by B-factor, non-linear NLS in spacefill K30 K20 K20 R29 R29 K30 apo holo

  7. CRABP II story Mutating K20, R29, K30 to ala abolishes nuclear import (Sessler & Noy 2005)

  8. CRABP II Results: RA causes CRABP II to accumulate in the nucleus This is due to nuclear import RA causes CRABP II to interact with importin  (DNS) conformational change upon RA binding results in a basic patch involving residues K20, R29, K30 Mutation of these residues abolishes nuclear import Conclusion: RA binding results in formation of a non-linear NLS

  9. K-FABP: Displays an even more complex behavior binds a wide spectrum of ligands with similar affinity nuclear localization response only to certain ligands activating (PPAR binding): linolenic acid non-activating: stearic acid WHY?

  10. K-FABP: 135AA, 1 disulfide K34 C R33 K24 N 1JJJ: NMR structure, holo with stearic acid

  11. R33 K24 K-FABP: Overlay of residues 20-38 of NMR models 1-20 of the human protein. There appears to be considerable conformational flexibility in K34 and especially K24. Suggests that dynamics are critical to the phenomenon. K34 K34 K24 R33

  12. K-FABP: How to answer the question: Why does K-FABP respond differently to different ligands? Solve the structure and query the dynamics in the presence of both activating and non-activating ligands Hypothesis: binding of an activating ligand results in the formation of or bias toward a non-linear NLS while a non-activating ligand does not

  13. Curiosity: What is the difference between iLBPs that do and don’t localize to the nucleus upon ligand binding?

  14. K-FABP: Action: Generate stable samples at NMR concentration Problem: The K-FABP samples are remarkably unstable a variety oflow salt buffers at multiple concentrations and pH’s result in sample aggregation

  15. K-FABP: 15N edited HSQC spectrum of stable sample 10mM HEPES pH 7.7, 40mM NaCl, 5mM DTT, 15°C

  16. K-FABP: Ongoing work: Spin system assignment 15N, TOCSY & NOESY 15N 13C, H(CC)(CO)NH and (H)CC(CO)NH Coming soon: Sequential assignment HNCA, HN(CA)CO, HNCO, HN(CO)CA (as needed) Backbone information 13C shift from random coil, HNCA 3JHN coupling constants,15N-HNHA Side chain information rotomer 1 angles, 3JH coupling 15N-HNHB Dipolor coupling 15N and 13C HSQC NOESY Dynamic analysis 15N - 1H NOESY

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